The long term goal of this research is to understand from a cellular and molecular perspective how specialized synaptic sites are generated, maintained and regulated between central neurons. Hippocampal neurons in low density culture provide an accessible model system to study excitatory glutamatergic and inhibitory GABAergic synapse development. The focus of this proposal is to use novel molecular, genetic and imagining tools to address two major issues: synapse dynamics and mechanisms of initiation; and cellular and molecular mechanisms of synaptic competition. Aim 1 will define changes in synapse stability with maturation by live imaging of synaptophysin-Cyan Fluorescent Protein and post-synaptic density protein PSD95-Yellow Fluorescent Protein. Aim 2 will combined these methods with fluorescent dye labeling of neuronal structure to determine the roles of axonal and dendritic filopodia in synapse initiation, as well as begin to test potential cell adhesion and kinase signaling mechanisms underlying synapse initiation. In Aims 3 and 4, model systems of hippocampal neurons in culture fostering synapse formation in environments of competitive activity will be generated, where the activity status of each pre- and post- synaptic element is known and the effects on arbor outgrowth and synapse formation and stability determined. Transmitter release or response will be manipulated by expression of tetanus toxin or dominant negative AMPA or NMDA type glutamate receptors or by genetic knockout of the GABA receptor anchoring protein gephyrin in subsets of neurons. These studies represent attacks of fundamental issues in developmental neurobiology generated through intellectual and practical interactions with Josh Sanes, Jeff Lichtman and Rachel Wong. The studies of mechanisms of synapse initiation (Aims 1-2) will lead toward promoting regeneration of connections after neurological insult and the studies of synaptic competition (Aims 3-4) will help to understand how early experience in infancy and childhood shape the connectivity of pathways in the brain.